An improved method for patch clamp recording and calcium imaging of neurons in the intact dorsal root ganglion in rats

J Neurosci Methods. 2008 Aug 15;173(1):74-82. doi: 10.1016/j.jneumeth.2008.05.023. Epub 2008 Jun 7.

Abstract

The properties of dorsal root ganglion (DRG) neurons have been mostly investigated in culture of dissociated cells, and it is uncertain whether these cells maintain the electrophysiological properties of the intact DRG neurons. Few attempts have been made to record from DRG neurons in the intact ganglion using the patch clamp technique. In this study, rat DRGs were dissected and incubated for at least 1h at 37 degrees C in collagenase (10mg/ml). We used oblique epi-illumination to visualize DRG neurons and perform patch clamp recordings. All DRG neurons exhibited strong delayed rectifier potassium current and a high threshold for spike generation (-15 mV) that rendered the cells very weakly excitable, generating only one action potential upon strong current injection (>300 pA). It is therefore possible that cultured DRG neurons, commonly used in studies of pain processing, may be hyperexcitable because they acquired "neuropathic" properties due to the injury induced by their dissociation. Electrical stimulation of the attached root produced an antidromic spike in the soma that could be blocked by intracellular hyperpolarization or high frequency stimulation. Imaging intracellular calcium concentration with Oregon Green BAPTA-1 indicates that antidromic stimulation caused a long-lasting increase in intracellular calcium concentration mostly near the cell membrane. This study describes a simple approach to examine the electrophysiological and pharmacological properties and intracellular calcium signaling in DRG neurons in the intact ganglion where the effects of somatic spike invasion can be studied as well.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cadmium / pharmacology
  • Calcium / metabolism*
  • Cells, Cultured
  • Diagnostic Imaging*
  • Dose-Response Relationship, Drug
  • Electric Stimulation / methods
  • Female
  • Ganglia, Spinal / cytology*
  • Male
  • Membrane Potentials / drug effects
  • Membrane Potentials / physiology
  • Membrane Potentials / radiation effects
  • Neurons / physiology*
  • Patch-Clamp Techniques / methods*
  • Potassium Channel Blockers / pharmacology
  • Rats
  • Rats, Sprague-Dawley
  • Tetraethylammonium / pharmacology

Substances

  • Potassium Channel Blockers
  • Cadmium
  • Tetraethylammonium
  • Calcium